GENE EXPRESSION AND HUMAN GENETICS

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Summary of work: In order to dissect the biochemical steps involved in genetic recombination we have chosen to focus on a key early step(s): homologous pairing and strand exchange between homologous parental DNAs. A fundamental problem in homologous recombination is how the search for homology between the two DNAs is carried out. In all current models a homologous recombination protein, such as the prototypical E. coli RecA protein, loads onto a single-strand DNA generated from one duplex DNA and scans another duplex to form a synaptic (pairing) complex. Eventually, DNA strands are exchanged and a new heteroduplex is formed. A novel technique developed by us in collaboration with Igor Panyutin of the Clinical Center, radioprobing of nucleoprotein structures with iodine-125, has allowed us to trace the spatial arrangement of the three DNA strands in the RecA protein mediated synaptic complex. The synaptic complex represents a poststrand exchange late intermediate in which the heteroduplex is located in the center and the outgoing strand forms a relatively wide and mobile helix intertwined with the heteroduplex. The structure implies that homology is recognized in the major groove of the duplex by the two extended DNAs. Efforts are underway to try to trap and characterize earlier intermediates in the pairing reaction, such as parallel DNA triplexes. In order to understand the mechanism and structures involved in greater detail we have endeavored to miniaturize the reaction. In the past, we have shown that short oligonucleotides can be used as the substrates. Recently, we have determined that a 20 amino acid peptide that includes loop L2 of RecA can promote the key reaction of the whole RecA protein: pairing (targeting) of a single stranded DNA to its homologous site on a duplex DNA. In the course of the reactions the peptide binds to both substrate DNAs, unstacks the single-stranded DNA, assumes a beta structure and self-assembles into a filamentous structure like RecA. It is possible that two DNAs align and pair on an extended beta-sheet of L2s in the whole RecA. In order to understand the function of L2 we have generated by site-directed mutagenesis all possible mutants of residues 193-212 in the whole RecA protein (380 mutants). The in vivo phenotype of these mutants with respect to recombination and UV and mitomycin resistance was determined. An analysis of these results suggested that L2 may be involved in most aspects of RecA function. As RecA is an ATP-dependent DNA binding protein and a DNA-dependent ATPase, we asked whether the loop might be directly involved in these allosteric interactions. We have been able to show that ATP, but not ADP, interacts with the arginine (Arg196) within L2 peptides and that this interaction induces the active beta-structure conformation of the peptides. Experiments with mutant RecA proteins indicate that Arg196 binds to both DNA and the gamma- phosphate of ATP and is essential for the cooperativity between DNA and ATP binding. We suggest a mechanism for ATP hydrolysis by RecA that is similar to those proposed for heterotrimeric G proteins. For example, that the role of DNA in the stimulation of the ATPase activity of RecA is similar to the role of the recently described RGS (Regulators of G protein Signaling) proteins in activating the GTPase of heterotrimeric G proteins and consists in stabilizing the highly mobile region involved in hydrolysis. Thus, other biopolymers in addition to proteins, such as DNA, can act to stimulate nucleotide hydrolysis by similar stereochemical regulatory mechanisms. Finally, we are investigating whether others domains of RecA are interacting with L2 and are responsible for regulating (positively or negatively) the efficiency of some of the biochemical activities of this loop. While homologous pairing and strand exchange are the earliest contacts between two parental DNAs, homologous recombination is initiated by DNA double-strand breaks (DSBs). The protein that catalyzes DSB formation in meiosis in the budding yeast, Saccharomyces cerevisae, is the product of the SPO11 gene. Disruption of this gene results in meiotic arrest, spore lethality and a lack of meiotic recombination. Spo11 homologs have been identified in other eukaryotes and archaebacteria resulting in the identification of a new family of proteins related to DNA topoisomerase IIs. We have identified a Drosophila melanogaster homolog to Spo11, DmSpo11, and isolated a cDNA from a Drosophila ovary cDNA library. We are studying structure-function relationships for this protein by examining the ability of chimeras of the fly and yeast proteins to rescue the spore-lethal meiotic defect in SPO11 deficient yeast. In addition, in collaboration with Brian Oliver (LCDB in NIDDK) transgenic flies are being generated to study the role of DmSpo11 may play in the recombination-less meiosis seen in male flies.